Restriction endonucleases are a group of enzymes originally found to be expressed in a wide variety of prokaryotic organisms. More recently they have also been found to be encoded in vital genomes. These enzymes catalyze the selective cleavage of DNA at generally short sequences, often unique to the individual enzyme. This ability to cleave makes restriction endonucleases indispensible tools in recombinant DNA technology. The increased commercial availability of the isolated enzymes has contributed in large part to the enormous expansion in the field of recombinant DNA technology over the last few years.
In bacteria, the restriction endonuclease provides a mechanism of defense against foreign DNA molecules (e.g., bacteriophage DNA) by virtue of its ability to distinguish and cleave only exogenous DNA, leaving endogenous bacterial DNA unaffected. Viral endonucleases possess the same discerning capabilities, but rather than providing a means for defense, this activity has presumably evolved to cripple the host's ability to replicate its own DNA and allows the virus to assume control of the host's replication machinery.
Bacteria and viruses which express restriction endonucleases necessarily possess the inherent ability to protect their own genome from cleavage by their endogenous endonuclease. The primary mechanism by which this is accomplished is by modifying the organisms own DNA by, for example methylating a base in the recognition sequence which prevents binding and cleavage by the endonuclease. Therefore, to insure viability, the genome of an organism which expresses a restriction endonuclease is almost always heavily modified, usually by methylation of cytosine or adenosine bases. The methylase enzyme which modifies the genome (itself a useful tool in molecular biology) acts in tandem with the endonuclease, either as part of an enzyme complex (restriction/modification complex) or as two distinct entities. Therefore, recognizing that an organism expresses an enzyme with endonuclease activity strongly suggests the expression of an associated modifying methylase enzyme (and vice versa) and this association has led to isolation and cloning of a number of commercially available restriction/modification enzymes for use in the laboratory as discussed below.
Endonucleases act by recognizing nucleotide sequences in a double stranded DNA molecule, binding to the sequence and cutting or cleaving the original DNA molecule into fragments. These fragments can then be isolated and propagated in a variety of ways to produce multiple copies of the desired sequence.
Although restriction endonucleases typically recognize and cut at a specific or defined sequence, in a number of cases the sequence specificity of isolated restriction endonucleases can be altered under appropriate reaction conditions (for example, by modifying glycerol concentration, ionic strength, pH, divalent cation concentration, or other conditions either individually or in combination). This altered specificity has been termed star (*) activity. As will be discussed below, the star activity of a given endonuclease can in some cases be exploited to great advantage in a number of molecular biological techniques.
One of the limitations in the use of restriction endonucleases exists when cleavage of a given sequence is required and no known endonuclease exists which is specific for that particular sequence. Therefore, the continued identification and isolation of unique restriction endonucleases and altered reaction conditions will allow for even more sophisticated manipulation of DNA in vitro.
A number of publications and patents describe the cloning of DNAs encoding restriction endonucleases. Included among theses publications is Kiss. A., et al., Nucleic Acid Research 13:6403-6421 (1985), which describes the cloned nucleotide sequence of the BsuRI restriction-modification system isolated from Bacillus subtillis. This system is specific for the sequence 5'-GGCC-3'and is defined by two gene products which are transcribed by different promoters. The methylase component of the system shows homology to the methylase from the BspRI and SPR restriction-modification systems.
Nwanko, D. O. and Wilson, G. G. Gene 64:1-8 (1988), describe the cloning and expression of the MspI restriction and modification genes isolated from Moraxella sp. This system recognizes the sequence 5'-CCGG-3' and both enzymes are functional in E. coli. Evidence indicates that these genes are transcribed in opposite directions, thus are probably under the control of different promoters.
Ashok, K. D., et al., Nucleic Acids Research 20:1579-1585 (1992), describe the purification and characterization of cloned MspI methyltransferase, over-expressed in E. coli. At low concentrations the enzyme exists as a monomer, but at higher concentrations it exists mainly as a dimer. Polyclonal antibodies to the enzyme cross-react with methyltransferase genes of other modification systems.
Brooks, J. E., et al. Nucleic Acids Research 19:841-850 (1991), characterizes the cloned BamHI restriction modification system from Bacillus subtilis. The two genes are divergently oriented and separated by an open reading frame which may serve as a transcriptional regulator in the native bacteria.
Slatko, B. E., et al. Nucleic Acids Research 15:9781-9796 (1987), describe the cloning, sequencing and expression of the TaqI restriction-modification system. These genes have the same transcriptional orientation, with the methylase gene 5' to the endonuclease gene. E. coli clones which carry only the endonuclease gene are viable even in the absence of the methylase gene. This is an unusual case possibly explained by the 65.degree. C. optimal temperature for TaqI restriction and the 37.degree. C. optimal temperature for E. coli growth.
Howard, K. A., et al., Nucleic Acids Research 14:7939-7951 (1986), describe the cloning of the DdeI restriction modification system from Desulfovibrio desulfuricans by a two step method wherein the methylase gene is first cloned and transformed into E. coli, followed by the cloning of the endonuclease gene and transformation of this second gene into the methylase-expressing bacteria. In order to maintain cell viability, high levels of methylase expression are required before the endonuclease gene can be introduced into the bacteria.
Ito, H., et al., Nucleic Acids Research 18:3903-3911 (1990), describe the cloning, nucleotide sequence and expression of the HincII restriction-modification system. The DNA was isolated from H. infuenzae Rc, with the two genes positioned in the same transcriptional orientation.
Shields, S. L., et al., Virology 76:16-24 (1990), describe the cloning and sequencing of the cytosine methyltransferase gene M.CviJI from the Chlorella virus IL-3A. The methylase recognizes the sequence (G/A)GC(T/C/G) and shows amino acid sequence homology with 5-methylcytosine methylases isolated from bacteria. DNA encoding the methylase was obtained from the viral genome which was propagated in the green alga host Chlorella.
Xia, Y., et al., Nucleic Acids Research 15:6075-6090 (1987), discovered that IL-3A virus infection of Chlorella-like green alga induces the expression of the DNA restriction endonuclease CviJI which has novel sequence specificity. This endonuclease recognizes the sequence PuGCPy (wherein Pu=purine and Py=pyrimidine) but does not cut the sequence PuG.sup.m CPy, where .sup.m C is 5-methylcytosine.
U.S. Pat. No. 5,137,823, issued Aug. 11, 1992, to Brooks, J. E., describes a two step method for cloning the BamHI restriction modification system wherein the methylase is cloned first and then introduced into a bacterial host. The endonuclease is then cloned and introduced into the methylase expressing bacteria. This two step procedure provides the host DNA protection from cleavage of the subsequently introduced endonuclease.
U.S. Pat. No. 5,200,333, ('333) issued Apr. 6, 1993, to Wilson, G. G., describes a method for cloning restriction and modification genes. Specifically this reference describes the cloning of the TaqI and HaeII systems from Thermus aquaticus and Haemophilus aegypticus, respectively. In this method, bacterial DNA was initially purified and digested, and the fragments were then cloned into a vector to produce a bacterial DNA library. The library was then transformed into E. coli and the cells were plated. Colonies were then scraped from the plate to form a primary cell library. Plasmid DNA from this cell library was purified and digested with the endonuclease of the two gene system. Bacteria which expressed the methylase gene had modified plasmid DNA which was protected from endonuclease activity, while plasmids from bacteria which lacked the intact methylase gene were digested. The resulting, undigested plasmid DNA was then transformed into another bacterial strain and the bacteria were plated. Surviving colonies were again harvested to give a secondary cell library and the entire procedure repeated. Plasmids which code for the complete restriction-modification system presumably survived each round of purification and were enriched. Bacteria which survive several rounds of enrichment were subsequently assayed for both methylase and endonuclease activity.
U.S. Pat. No. 5,196,331, ('331) issued Mar. 23, 1993, to Wilson, G. G. and Nwanko, D., describes a method for cloning the MspI restriction and modification genes. This patent describes a method identical to that of U.S. Pat. No. 5,200,333 ('333). '331 is a continuation-in-part of, and '333 is a continuation of U.S. Ser. No. 707,079 (now abandoned).
As mentioned above, Chlorella virus IL-3A encodes a unique restriction endonuclease called CviJI (Xia et al. Nucleic Acids Res. 15:6075-6090 (1987)). IL-3A is a large, polyhedral, plaque-forming phycodnavirus (Francki, R. I. B., et al. Arch. Virol. suppl. 2. Springer-Verlag, Vienna (1991)) that replicates in unicellular, eukaryotic green algae, Chlorella strain NC64A (Schuster, A. M., et al. Virology 150:170-177 (1986)). The double-stranded DNA genome of IL-3A is approximately 330 kbp (Rohozinski et al., Virology 168:363-369 (1989)) and contains 9.7% methylated cytidine (Van Etten, J. L. et al., Nucleic Acids Res. 13:3471-3478 (1985)). The cognate methyltransferase of CviJI, M.CviJI, methylates (A/G)GC(T/C/G) sequences and, has been cloned and sequenced (Shields, S. L. et al., Virology 176:16-24 (1990)).
CviJI is an unusual restriction endonuclease which is capable of digesting DNA at a two base or three base recognition sequence, depending on the reaction conditions. CviJI normally recognizes the sequence PuGCPy and cleaves between the G and C residues to leave blunt ends (Xia et al. Nucleic Acids Res. 15:6075-6090 (1987)). Under relaxed or star conditions (in the presence of 1 mM ATP and 20 mM DTT) the specificity of CviJI can be altered to cleave DNA more frequently. This activity is referred to as CviJI*, for star or altered specificity. However, CviJI* activity is not observed under conditions which favor star activity of other restriction endonucleases.
The use of a two/three base recognition endonuclease, such as CviJI, to improve numerous conventional molecular biology applications as well as permitting novel applications has been described in co-pending U.S. patent application Ser. No. 08/036,481, filed on Mar. 24, 1993. The application discloses methods for generating sequence-specific oligonucleotides from DNA without prior knowledge of the nucleic acid sequence of such DNA, and to methods for cloning and labeling DNA after restriction digestion by a two base recognition endonuclease. The application also teaches methods for generating quasi-random fragments of DNA, methods for cloning, labeling, and sequencing DNA, as well as epitope mapping of proteins. The ability to generate numerous oligonucleotides with perfect sequence specificity or quasi-random distributions of DNA fragments such as is possible with CviJI* has important implications for a number of conventional and novel molecular biology procedures.
Infection of Chlorella species NC64A with the IL-3A virus produces sufficient CviJI restriction endonuclease (CviJI) for research purposes. However, production of commercially useful amounts of CviJI is limited with this system due to the slow growth of Chlorella algae, the large number of contaminating nucleases associated with the virus, and the small yield of enzyme obtained after purification. In addition, biochemical and biophysical characterization of the enzyme, such as molecular weight determination, are difficult from the native source. Because of these limitations it would be useful to clone the gene for CviJI in order to provide an adequate large scale source of enzyme for use as a molecular biological reagent.